1 DNA Structure The building blocks of nucleic acids are nucleotides, each composed of: –a 5-carbon sugar called deoxyribose –a phosphate group (PO 4 ) –a nitrogenous base adenine, thymine, cytosine, guanine, uracil

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3 Nucleosides Nucleosides: nitrogenous base linked to specific sugar –RNA: adenosine, guanosine, cytidine, uridine –DNA: deoxyadenosine, deoxyguanosine, deoxycytidine, (deoxy)thymidine /.../Nucleosides.gif DNA nucleoside RNA nucleoside

4 Nucleotides The nucleotide structure consists of –the nitrogenous base attached to the 1’ carbon of deoxyribose –the phosphate group attached to the 5’ carbon of deoxyribose –a free hydroxyl group (-OH) at the 3’ carbon of deoxyribose

5 Nucleotides Subunits of DNA and RNA –Nucleosides linked to phosphate group via ester bond –“dNTP’s”: DNA –“rNTP’s”: RNA

6 DNA Structure Nucleotides are connected to each other to form a long chain phosphodiester bond: bond between adjacent nucleotides –formed between the phosphate group of one nucleotide and the 3’ –OH of the next nucleotide The chain of nucleotides has a 5’ to 3’ orientation.

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8 DNA structure determination Chargaff's Rules –Erwin Chargaff determined that amount of adenine = amount of thymine amount of cytosine = amount of guanine

9 DNA Structure The double helix consists of: –2 sugar-phosphate backbones –nitrogenous bases toward the interior of the molecule –bases form hydrogen bonds with complementary bases on the opposite sugar-phosphate backbone Adenine pairs with Thymine (2 H bonds) Cytosine pairs with Guanine (3 H Bonds)

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11 DNA Structure The two strands of nucleotides are antiparallel to each other –one is oriented 5’ to 3’, the other 3’ to 5’ The two strands wrap around each other to create the helical shape of the molecule.

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13 Types of DNA Structures Three forms of DNA –A form: right handed helix –B form: the most likely biological conformation, right handed helix –Z form: form a left handed helix;

14 Chemical Properties of DNA Factors that affect DNA structure –Temperature: denaturation (can be reversible) –pH: high pH can denature DNA –Salt concentration: lowering salt concentration can denature DNA –Chemicals: sodium hydroxide, formamide can also denature DNA

15 DNA Replication Matthew Meselson & Franklin Stahl, 1958 investigated the process of DNA replication considered 3 possible mechanisms: –conservative model –semiconservative model –dispersive model

16 At the 0 time point: all the DNA had heavy 15N nitrogen After 1 round: the DNA was a hybrid molecule, with an intermediate location After 2 rounds: two molecules were seen: one that was hybrid, and one that was the lighter 14N DNA molecule. Conclusion: Semiconservative replication

17 DNA Replication Meselson and Stahl concluded that the mechanism of DNA replication is the semiconservative model. Each strand of DNA acts as a template for the synthesis of a new strand.

18 DNA Replication DNA replication includes: –initiation – replication begins at an origin of replication –elongation – new strands of DNA are synthesized by DNA polymerase –termination – replication is terminated differently in prokaryotes and eukaryotes

19 Prokaryotic DNA Replication The chromosome of a prokaryote is a circular molecule of DNA. Replication begins at one origin of replication and proceeds in both directions around the chromosome. --origins of replications usually are rich in Adenine and Thymine

20 Enzymes of Prokaryotic DNA Replication The double helix is unwound by the enzymes helicase, DNA topoisomerase,and DNA gyrase –SSBP (single stranded binding protein) helps keep strands separated DNA polymerase III (pol III) is responsible for most of DNA synthesis –adds nucleotides to the 3’ end of the daughter strand of DNA; DNA synthesis is from 5' to 3' –Requires RNA primers as a guide for synthesis RNA primers are made by the enzyme primase

21 Enzymes of Prokaryotic DNA Replication DNA polymerase I: involved in proofreading and DNA repair DNA ligase: involved in connected ends of replicated DNA together

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23 Prokaryotic DNA Replication leading strand is synthesized continuously (in the same direction as the replication fork) lagging strand is synthesized discontinuously creating Okazaki fragments

24 Eukaryotic DNA Replication The larger size and complex packaging of eukaryotic chromosomes means they must be replicated from multiple origins of replication. The enzymes of eukaryotic DNA replication are more complex than those of prokaryotic cells.

25 Eukaryotic DNA Replication Synthesizing the ends of the chromosomes is difficult because of the lack of a primer. With each round of DNA replication, the linear eukaryotic chromosome becomes shorter.

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27 Eukaryotic DNA Replication telomeres – repeated DNA sequence on the ends of eukaryotic chromosomes –produced by telomerase telomerase contains an RNA region that is used as a template

28 DNA organization in cells Prokaryotes –DNA is circular –DNA not usually associated with proteins –Some have plasmids: small circular molecules of DNA outside of the main genomic DNA Eukaryotes –Three locations for DNA: nucleus, mitochondria, chloroplasts –Nuclear DNA is linear, associated with protein –Organelle DNA is circular, not associated with proteins

29 Eukaryotic Nuclear DNA organization Nucleosome: DNA associated with histone protein Chromatin: collection of nucleosome and linker DNA Chromosome: condensed chromatin –Ends of chromosomes are called telomeres (very repetitive sequences)

30 What is a genome? Genome: the entire collection of DNA for a given organism and/or organelle –Bacterial genomes: sum total of all DNA (not including plasmids) –Nuclear genomes: sum total of all DNA in nucleus –Mitochondrial, chloroplast genome

31 Organization of DNA DNA reassociation kinetics –Allow DNA for a given species to denature (usually by heat) –Time how long it takes for the DNA to renature –Repetitive sequences renature faster than nonrepetitive(unique) sequences –Complexity: more complex genomes have more unique sequences

32 Types of DNA in genomes Classification based on reassociation kinetics Three classes –Highly repetitive: About 10-15% of mammalian DNA –Moderately repetitive: Roughly 25-40% of mammalian DNA. –Single copy (or very low copy number): This class accounts for 50-60% of mammalian DNA (thought to be regions that encode mRNA and/or protein—genes)